Production of Commercially Suitable

Production of Commercially Suitable

Pectin methylesterase and Polyphenol oxidase
from Agro-industrial Wastes

By

Şebnem ŞİMŞEK

A Dissertation Submitted to the

Graduate School in Partial Fulfillment of the

Requirements for the Degree of
MASTER OF SCIENCE

Department : Food Engineering

Major : Food Engineering

İzmir Institute of Technology

İzmir, Turkey
July, 2004

We approve the thesis of Şebnem ŞİMŞEK

Date of Signature
23.07.2004

Assoc. Prof. Dr. Ahmet YEMENİCİOĞLU

Supervisor

Department of Food Engineering
23.07.2004

Prof. Dr. Şebnem HARSA

Department of Food Engineering

23.07.2004

Assist. Prof. Dr. Oğuz BAYRAKTAR

Department of Chemical Engineering

23.07.2004

Prof. Dr. Şebnem HARSA

Head of Food Engineering Department

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor Assoc. Prof. Ahmet Yemenicioğlu for his guidance, supervision, encouragement, and support at all steps of this study.

I am also grateful to my family for their support, encouragement, and understanding.

ABSTRACT

In this study, some simple and effective extraction and/or partial purification procedures were developed to obtain pectin methylesterase (PME) and polyphenol oxidase (PPO) enzymes from orange peels and mushroom stems, respectively. Also, some characteristics of enzymes were investigated and their stable preparations were obtained in liquid or lyophilized forms. Valencia orange peels contain considerable PME activity (300-350 mL NaOH/min/100 g) that is quite stable during season for at least 5 months. The enzyme was ionically bound to cell walls and can not be extracted by homogenization with water. However, the addition of suitable amounts of NaCl (10 g /100 g extraction mixture) to pellet, obtained by homogenization of peels several times with water, and 30 min mixing (at 200 rpm) may be effectively used to extract the enzyme. The PME in orange peels contains almost the same amount of heat stable and heat labile fractions and the enzyme can not be activated by mild heating. A slight activation (almost 20 %) may be achieved by adding 1 mM CaCl₂ to enzyme extracts. However, at higher concentrations the addition of CaCl₂ was inhibitory. The PME activity in extracts, stabilized by use of 0.1 % Na-benzoate and 0.1 % K-sorbate, is stable almost 5 months at + 4 ^oC (maintains > 90 % of its activity). Thus, the commercial preparations of enzyme may be obtained in liquid form. The extracted PME was successfully used to prepare edible films from citrus pectin.

For the extraction of PPO, on the other hand, mushroom stems were first processed to acetone powder. The acetone powders were then extracted with Na-phosphate buffer and partially purified with ammonium sulfate (90 % saturation) or acetone precipitation (2-fold). Following dialysis, the recoveries and purification folds obtained from the partial purification of monophenolase activity of PPO from the same acetone powder were 74-86 % and 3.4-4.3 and 55-67 % and 5.4-6.2 for ammonium sulfate and acetone precipitations, respectively. Thus, it appears that the ammonium sulfate precipitation gives a higher yield but lower purity. The monophenolase activity of partially purified PPO is heat labile and showed inactivation above 45 ^oC. The enzyme exhibited a pH optimum between pH 6.0 and 8.0. The pH stability of enzyme was maximal at pH 7.0 and 8.0. However, at pH 4.0 the enzyme lost most of its activity after 24 h incubation. The optimum temperature of enzyme was found as 40 ^oC. The monophenolase activity of PPO enzyme showed no stability in acetone powders at + 4 ^oC. However, it showed good stability at -18 ^oC for two months with retention of 60-70 % of its activity. The PPO partially purified with ammonium sulfate precipitation and dialysis, and lyophilized by using dextran or saccharose as supporting materials also retained its monophenolase and diphenolase activities for three months at -18 ^oC. The effect of lyophilization with dextran on temperature stability of enzyme was insignificant. However, lyophilization with dextran reduced the pH stability of monophenolase activity at 4.0 moderately. In addition to its monophenolase activity on tyrosine and diphenolase activity on L-DOPA, PPO lyophilized with dextran can also use phloridzin as substrate. Thus, it appears that the enzyme may be used in different food applications including the production of antioxidants and colorants, modification of proteins, fermentation of cocoa and black tea, etc.

ÖZ
Bu çalışmada pektin metilesteraz (PME) ve polifenol oksidaz (PPO) enzimlerinin sırasıyla portakal kabukları ve mantar saplarından eldesi için pratik ve etkili olabilecek ekstraksiyon ve kısmi saflaştırma prosedürleri geliştirilmiştir. Ayrıca enzimlerin bazı karakteristikleri de incelenmiş ve sıvı veya liyofilize haldeki stabil preparatları da hazırlanmıştır. Çalışmada Valencia portakal kabuklarında kayda değer miktarda PME aktivitesi tesbit edilmiş (300-350 mL NaOH/dak/100gr) ve bu aktivitenin sezon boyunca en az 5 ay stabilitesini koruduğu belirlenmiştir. Portakal kabuklarında bulunan enzim, hücre duvarına iyonik olarak bağlı olup su ile ekstrakte edilememekte, ancak buna karşın kabukların su ile birkaç kez homojenizasyonu ve filtrasyonuyla elde edilen kitleden uygun oranda NaCl ilavesi (10 gr/100 gr ekstraksiyon karışımı) ve karıştırma ile (200 rpm’de 30 dak) etkili bir şekilde ekstrakte edilebilmektedir. Portakal kabuklarındaki PME aktivitesinin yaklaşık yarısı ısıya dirençli, yarısı da ısıya duyarlı enzim fraksiyonlarından oluşmakta olup, ısının enzim üzerinde aktive edici bir özelliği belirlenememiştir. Enzim ekstraktına 1 mM CaCl₂ ilavesi ile zayıf bir aktivasyon (yaklaşık % 20) sağlanabilmekte, ancak buna karşın yüksek konsantrasyonlardaki CaCl₂ ilavesi inhibe edici özellik göstermektedir. % 0.1 Na-benzoat ve % 0.1 K-sorbat ile stabilize edilmiş ekstraktlardaki PME aktivitesi + 4 ^oC’de yaklaşık 5 ay stabildir (% 90’ın üzerinde aktivitesini korumaktadır). Buna göre portakal kabuğundan elde edilen PME’nin ticari preparatlarının sıvı formda hazırlanmasında herhangi bir sakınca bulunmamaktadır. Bu çalışmada hazırlanmış olan PME preparatı narenciye pektininden yenilebilir film üretiminde başarılı bir şekilde kullanılmıştır.

Diğer yandan, PPO’nun ekstraksiyonu için öncelikle mantar sapları aseton tozuna işlenmiştir. Aseton tozları daha sonra Na-fosfat tamponu ile ekstrakte edilmiş ve % 90 amonyum sülfat çöktürmesi ya da 2–kat aseton çöktürmesi ve bunu takip eden diyaliz işlemi ile kısmi olarak saflaştırılmıştır. Aynı aseton tozundaki PPO’nun monofenolaz aktivitesinin, amonyum sülfat veya asetonla çöktürme yoluyla kısmi saflaştırılması ile elde edilen geri kazanım ve saflık katsayıları sırasıyla % 74-86 ve 3.4-4.3 ve % 55-67 ve 5.4-6.2’dir. Buna göre amonyum sülfat çöktürmesinin daha yüksek verime karşın daha düşük saflık sağladığı görülmektedir. Kısmi olarak saflaştırılmış PPO enziminin monofenolaz aktivitesinin ısıl direnci düşük olup inaktivasyonu 45 ^oC’ın üzerinde başlamaktadır. Enzim, pH 6.0 ve 8.0 arasında optimum aktivite göstermiş olup pH 7.0 ve 8.0’deki stabilitesi maksimumdur. Ancak buna karşın enzim, pH 4.0’te 24 saat inkübasyon sonucunda aktivitesinin büyük kısmını kaybetmiştir. Enzimin optimum sıcaklığı 40 ^oC olarak belirlenmiştir. PPO enziminin monofenolaz aktivitesi, aseton tozu halinde + 4 ^oC’de stabilite göstermemiş, ancak buna karşın - 18 ^oC’de iki ay süreyle % 60-70 oranında aktivitesini korumuştur. Ayrıca amonyum sülfat ve diyaliz ile kısmi saflaştırılmış ve destek maddesi olarak dekstran ve sakkaroz eklenerek liyofilize edilmiş PPO, –18 ^oC’de 3 ay süresince monofenolaz ve difenolaz aktivitesini korumuştur. Dekstranla liyofilizasyonun enzimin ısıl stabilitesi üzerinde herhangi bir etkisi belirlenememiştir. Ancak buna karşın dekstranla liyofilizasyon, pH 4.0’deki monofenolaz aktivitesinin stabilitesini kısmen azaltmıştır. Tirozin üzerindeki monofenolaz aktivitesi ve L-DOPA üzerindeki difenolaz aktivitesinin yanısıra dekstranla liyofilize edilmiş PPO, floridzin’i de substrat olarak kullanabilmektedir. Bu sonuç enzimin antioksidan ve renk maddelerinin üretimi, proteinlerin modifikasyonu, kakao ve siyah çayın fermantasyonu gibi farklı gıda uygulamalarında kullanılabileceğini göstermektedir.

TABLE OF CONTENTS

LIST OF FIGURES	xii
LIST OF TABLES	xiv
CHAPTER 1. INTRODUCTION	1
CHAPTER 2. ENZYME PRODUCTION FOR INDUSTRIAL APPLICATIONS	3
2.1. Extraction of Enzymes	3
2.1.1. Cell disruption methods	5
2.1.1.1. Mechanical methods	5
2.1.1.2. Nonmechanical methods	5
2.2. Clarification of Enzyme Extracts	6
2.2.1. Centrifugation	6
2.2.2. Filtration	6
2.2.3. Flocculation and flotation	7
2.3. Concentration of Enzymes	7
2.3.1. Addition of a dry matrix polymer	7
2.3.2. Freeze-drying (Lyophilization)	7
2.3.3. Ultrafiltration	8
2.3.4. Precipitation	9
2.3.4.1. Precipitation by increasing the ionic strength (salting out)	9
2.3.4.2. Precipitation by decreasing the ionic strength (salting in)	10
2.3.4.3. Precipitation by organic solvents	10
2.3.4.4. Precipitation by alteration of pH	11
2.3.4.5. Precipitation by organic polymers	11
2.3.4.6. Precipitation by denaturation	11
2.3.5. Aqueous two-phase partitioning	12
2.3.6. Removal of salts and exchange of buffers	12
2.3.6.1. Dialysis	12
2.3.6.2. Diafiltration	13
2.3.6.3. Gel filtration	13
2.4. Purification	13
2.5. Product Formulation	14
CHAPTER 3. PECTIN METHYLESTERASE	16
3.1. Pectin methylesterase and Other Pectinases	16
3.2. Sources of PME	16
3.3. The Effects of PME on Food Quality	17
3.4. Industrial Applications of PME	19
3.4.1. Clarification of fruit juices	19
3.4.2. Firming of fruits and vegetables before processing	21
3.4.3. Modification of pectin	21
3.4.4. Production of low sugar jams, jellies, and compotes	22
3.4.5. Other applications	22
CHAPTER 4. POLYPHENOL OXIDASES	24
4.1. Polyphenol oxidases	24
4.2. Substrates of PPO	26
4.3. Sources and Some Characteristics of PPO	27
4.4. The Effects of PPO on Food Quality	29
4.5. Industrial Applications of PPO	29
4.5.1. Enzymatic cross-linking of proteins or polysaccharides	30
4.5.2. Production of flavonoid-derived colorants and antioxidants	30
4.5.3. The removal of haze forming polyphenols from beverages	31
4.5.4. Oxygen scavenging and removal of undesirable phenolics from food	32
4.5.5. Removal of undesirable phenolics from wastewaters	32
4.5.6. Analytical and clinical applications of PPO	33
4.5.6.1. Production of biosensors	33
4.5.6.2. Clinical applications	33
CHAPTER 5. MATERIALS AND METHODS	35
5.1. Materials	35
5.2. Methods	35
5.2.1. Methods related to PME enzyme	35
5.2.1.1. PME extraction	35
5.2.1.2. Determination of PME activity	37
5.2.1.3. Effect of mild heating on PME activity	37
5.2.1.4. Effect of CaCl2 on PME activity	38
5.2.1.5. Preparation of a commercial PME preparation and test of its stability	38
5.2.1.6. Test of obtained PME in the preparation of edible pectin films	38
5.2.2. Methods related to PPO enzyme	39
5.2.2.1. Acetone powder preparation	39
5.2.2.2. PPO extraction	39
5.2.2.3. Ammonium sulphate precipitation	39
5.2.2.4. Acetone precipitation	39
5.2.2.5. Determination of PPO activity	40
5.2.2.6. Characterization studies	40
5.2.2.7. Storage stability of PPO in acetone powders	41
5.2.2.8. Preparation of commercial PPO preparations and test of their storage stabilities	41
5.2.2.9. The effect of lyophilization with dextran on temperature and pH stability of PPO	41
5.2.3. Determination of protein content	42
CHAPTER 6. RESULTS AND DISCUSSION	43
6.1. The Results Obtained for PME Enzyme	43
6.1.1. Change of PME activity in orange peels during season	43
6.1.2. Effect of different extraction procedures on PME activity	44
6.1.3. Effect of NaCl concentration on PME activity extracted from orange peels	45
6.1.4. Effect of extraction period on PME activity extracted from orange peels	46
6.1.5. Effect of mild heating on PME activity	47
6.1.6. Effect of CaCl2 on PME activity	48
6.1.7. Stability of the prepared PME during storage	49
6.1.8. Test of obtained PME in the preparation of edible pectin films	50
6.2. The Results Obtained for PPO Enzyme	52
6.2.1. Monophenolase and diphenolase activities of PPO	52
6.2.2. Distribution of PPO in mushrooms	53
6.2.3. Partial purification of PPO	54
6.2.4. Characterization of monophenolase activity	56
6.2.5. Stability of monophenolase activity in acetone powders	60
6.2.6. Stability of the prepared PPO during storage	61
6.2.7. The effect of lyophilization with dextran on temperature and pH stability of PPO	63
6.2.8. The ability of the prepared PPO to oxidize phloridzin	64
CHAPTER 7. CONCLUSIONS	66
REFERENCES	68